The long-term goals of the proposed research are: 1) to understand the role of the plant hormone indole-3-acetic acid (IAA) in plant growth and development using mutants of Arabidopsis; 2) to identify and characterize the proteins required for auxin action. This proposal describes experiments which address the function of three auxin-resistance genes called AXR1, AXR3,and AXR4. The AXR1 gene has been cloned and encodes a novel protein with similarity to ubiquitin-activating enzyme (El). We intend to continue our molecular analysis of this gene, including the characterization of gene structure and regulation. The cellular location of the Axr1 protein will be established by cell fractionation studies as well as immunofluorescence microscopy. The potential role of the Axr1 protein in the ubiquitin pathway will be assessed in a series of in vitro studies using purified protein. To examine the role of ubiquitination in plant hormone action, we will characterize ubiquitin-protein conjugates in the axr1 mutants. The effects of ectopic expression of the AXR1 gene on hormone sensitivity and plant development will be determined. To identify proteins which interact with the Axr1 protein we have isolated a series of mutations which suppress the axr1 mutants. These mutations (called suppressor of auxin resistance, sar) will be characterized. We expect that the analysis of these interacting genes will provided important information on the function of the AXR1 gene as well as the SAR loci. We also propose to study two additional auxin-resistance genes called AXR3 and AXR4. The phenotypes of axr3 and axr4 mutants suggest that both genes encode proteins required for hormone action. We intend to clone and characterize both of these loci. We anticipate that our studies will lead to significant new insight into the mechanism of plant hormone action. In addition, our analyses of the AXR1 gene may contribute to an understanding of regulatory mechanisms in all eukaryotes. The ubiquitin pathway has been implicated in diverse cellular processes, including cell cycle control, DNA replication, gene expression, viral infection and oncogenesis. Our studies indicate that changes in an E1-like protein result in a reduction in plant hormone sensitivity. Thus, further analysis of this protein may lead to the discovery of a new regulatory function for ubiquitin.